香港海鸥菌HdeA蛋白的原核表达及活性研究

Prokaryotic expression of the HdeA protein fromLaribacter hongkongensis

目的表达香港海鸥菌(Laribacter hongkongensis)分子伴侣蛋白HdeA并鉴定其保护功能,为解析其抗酸机制奠定基础。方法通过与相关蛋白进行同源性比较,在香港海鸥菌中找出编码HdeA的基因。以HdeA基因序列为基础,设计特异性引物,PCR扩增HdeA基因,并将其连接到表达载体pET28a,构建的重组质粒pET28a-HdeA转化大肠埃希菌BL21(DE3),得到重组菌BL/pET28a-HdeA,用IPTG诱导表达目的蛋白并利用镍柱亲和层析纯化HdeA蛋白,采用光散射法分析HdeA蛋白的保护功能。结果通过Blast分析,从香港海鸥菌中找到编码HdeA的基因。以合成的特异性引物扩增出约315bp的HdeA片段,连接到原核表达载体pET28a后转化大肠埃希菌BL21,经IPTG诱导成功表达出HdeA蛋白,其分子质量单位为14.1ku,与理论值相符合。经His-tag亲和层析纯化,获得纯度较高的重组HdeA蛋白。光散射法检测分析HdeA蛋白能够减轻酸性条件下ADH蛋白聚集。结论纯化的重组HdeA蛋白在体外具有抗酸功能,为研究香港海鸥菌的抗酸机制奠定了基础。

Objective The aim of this study was to identify the HdeA gene fromLaribacter hongkongensis and evaluate its counteraction of acids in order to understand the mechanisms of acid tolerance in L.hongkongensis. Methods A protein homology search with blast was used to analyze the HdeA gene fromL.hongkongensis.The coding sequence of the HdeA gene was inserted into a pET28 avector to generate a pET28a-HdeA recombinant plasmid.This plasmid was transformed into E.coli BL21（DE3）to create a transformed strain,BL/pET28a-HdeA.The HdeA protein was induced with IPTG and purified with Ni 2＋-affinity chromatography.The protective role of HdeA protein was measured with a light scattering assay. Results Bioinformatics was used to identify the HdeA gene of L.hongkongensis.A 315-bp HdeA fragment was amplified from the genome of L.hongkongensis and was cloned into a pET28 aexpression vector.The recombinant vector pET28a-HdeA was transformed into E.coli BL21.The HdeA protein was induced with IPTG and was successfully purified.Acid-induced alcohol dehydrogenase（ADH）aggregation was attenuated by the HdeA protein.Conclusion Results indicated that the purified HdeA protein counteracts acids in vitro,shedding light on the mechanism of acid stress tolerance in L.hongkongensis.